1. Field of the Invention
The present invention relates to the detection of a target nucleic acid sequence by a cyclic exonucleolytic reaction.
2. Description of the Related Art
A target nucleic acid amplification process is prevalently involved in most of technologies for detecting target nucleic acid sequences. Nucleic acid amplification is a pivotal process for a wide variety of methods in molecular biology, such that various amplification methods have been proposed. For example, Miller, H. I. et al. (WO 89/06700) amplified a nucleic acid sequence based on the hybridization of a promoter/primer sequence to a target single-stranded DNA (“ssDNA”) followed by transcription of many RNA copies of the sequence. Other known nucleic acid amplification procedures include transcription-based amplification systems (Kwoh, D. et al., Proc. Natl. Acad. Sci. U.S.A., 86:1173 (1989); and Gingeras T. R. et al., WO 88/10315).
The most predominant process for nucleic acid amplification known as polymerase chain reaction (hereinafter referred to as “PCR”) is based on repeated cycles of denaturation of double-stranded DNA, followed by oligonucleotide primer annealing to the DNA template, and primer extension by a DNA polymerase (Mullis et al. U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159; Saiki et al., (1985) Science 230, 1350-1354).
PCR-based techniques have been widely used not only for amplification of a target DNA sequence, but also for scientific applications or methods in the fields of biological and medical research, such as reverse transcriptase PCR (RT-PCR), differential display PCR (DD-PCR), cloning of known or unknown genes by PCR, rapid amplification of cDNA ends (RACE), arbitrary priming PCR (AP-PCR), multiplex PCR, SNP genome typing, and PCR-based genomic analysis (McPherson and Moller, (2000) PCR. BIOS Scientific Publishers, Springer-Verlag New York Berlin Heidelberg, N.Y.).
In addition, a variety of real-time PCR procedures have been proposed for detecting target nucleic acids. The real-time PCR methods have been highlighted in the senses that they can amplify and detect target sequences in a real-time manner. The real-time PCR procedures generally use labeled-primers or labeled-probes. The illustrative examples of the real-time PCR procedures include TaqMan™ probe method (U.S. Pat. No. 5,210,015), Self-quenching probe method (U.S. Pat. No. 5,723,591), Molecular Beacon method (Tyagi et al, Nature Biotechnology v. 14 Mar. 1996), and Lion method (U.S. Pat. No. 6,248,526).
In TaqMan™ probe method, probes are designed to hybridize to an internal region of a PCR product. While the polymerase replicates a template on which a TaqMan probe is bound, the 5′ to 3′ exonuclease activity of the polymerase cleaves the probe. This separates the fluorescent and quenching dyes and FRET no longer occurs (Parashar et al, Indian J Med Res 124: 385-398(2006)). According to TaqMan™ probe method, the 5′ to 3′ exonuclease activity of the polymerase triggers exonucleolytic exoncleolytic reactions only when the polymerase binds to upstream primers or their extended products. When the primer and the probe are designed to be in a proximal position, the 5′ to 3′ exonuclease activity bound to the primer cleaves the probe close to the primer with no polymerization reactions, called as a polymerization-independent cleavage. Meanwhile, where the primer and the probe are in a distal position, the polymerase extends the primer and its 5′ to 3′ exonuclease activity bound to the primer extended product cleaves the probe, called as a polymerization-dependent cleavage (U.S. Pat. No. 5,210,015, column 6, line 41 to column 7, line 10).
The Self-quenching probe method or Molecular beacon method uses dual-labeled probes having a sequence hybridizable with an internal region of a PCR product. In both methods, upon hybridization with target sequences, probes generate fluorescent signals by unquenching signals.
The Lion method uses a labeled primer deliberately mismatched in at least one nucleotide at the 3′ end of the primer. The labeled primer is incubated with a sample under conditions sufficient to allow hybridization and the sample is subsequently exposed to nucleic acid polymerase having a 3′ to 5′ proofreading activity, thereby releasing the label or part of the label system.
In light of signal generation, the TaqMan™ probe method has some disadvantages associated with requirement for primers for probe cleavage. For example, the requirement for primers as well as probes becomes a serious obstacle for multiple target detection. The Self-quenching probe method or Molecular beacon method produce no signal accumulation over cycles of PCR reactions, which is considered ineffective in signal generation. In addition, both methods based on hybridization have to detect signals under conditions for hybridization with target sequences. Such signal detection protocol is responsible for their poor applicability to various conditions.
In the meantime, the real-time PCR methods including those described above basically require amplification primers for amplification of target sequences as well as target signals for simultaneous detection of the amplified products. Therefore, they are called homogeneous assays. However, the requirement of amplification primers in the real-time PCR methods provides a limitation in the application for detection of multiple target sequences such as false positive signals and difficulties in oligonucleotides (primer and probe) selection and reaction condition optimization.
Therefore, it could be appreciated that a new approach to amplify signals with no help of simultaneous target amplification may enable to detect multiple target sequences without such problems accounted in the conventional real-time PCR methods.
Throughout this application, various patents and publications are referenced, and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.